Alpha Polypeptide Chains Research Articles

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-cell-derived I-A.

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that alpha, beta and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived Ia antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed alpha and beta chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between alpha and beta chains.

Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3.

Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains alpha and beta polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alpha 1 beta 1 structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.

Biochemical evidence for multiple I-E Ia molecules.

Sequential immunoprecipitation and isoelectric focusing analyses with monoclonal I-E-specific antibodies presented in this paper indicate the existence of multiple I-E molecules. In sequential immunoprecipitations with 13-4 (anti-Ia.7) and 17-3-3 (anti-Ia.22) monoclonal antibodies, 17-3-3 only partially cleared I-E molecules immunoprecipitated by 13-4. Similarly, 13-4 monoclonal antibody only partially cleared I-E molecules precipitated by 17-3-3 monoclonal antibody. These results suggested a minimum of three I-E molecules. One I-E molecule expresses both 13-4 and 17-3-3 determinants, a second I-E molecule expresses only 17-3-3 determinants, and a third I-E molecule expresses only 13-4 determinants. Isoelectric focusing analyses of I-E molecules immunoprecipitated by 13-4 and 17-3-3 showed differences in both Ae beta polypeptide chains and E alpha polypeptide chains. The sequential immunoprecipitation and isoelectric focusing analyses presented in this paper can be explained by a model in which there are at least two separate Ae genes being encoded within the I-A subregion and two separate E alpha genes being encoded within the I-E subregion. The 17-3-3 monoclonal antibody would recognize a determinant on only one of two Ae beta polypeptide chains and the 13-4 monoclonal antibody would recognize a determinant on only one of two E alpha polypeptide chains.

Role of Ia antigens in graft vs. host reactions. II. Molecular and functional analysis of T cell alloreactivity by the characterization of host Ia antigens on alloactivated donor T cells.

Graft vs. host response (GVHR)-activated donor T cells bind to stimulatory host cell-derived Ia antigens. Radioimmune cell-binding assays demonstrate that activated donor T cells acquire both host I-A and I-E alloantigens on their surface. Approximately threefold to fivefold less I-E products than I-A products are transferred. Immunoprecipitation and one-dimensional and two-dimensional gel electrophoresis analyses show that radioiodinated alpha and beta polypeptide chains of both I-A and I-E-encoded host Ia molecules may be transferred in an apparently structurally unaltered form from host cells to donor cells. Biosynthetic studies indicate that [35S]methionine-labeled activated donor T cells do not synthesize Ia antigens of the donor haplotype. Functional analyses with fluorescence-activated cell sorter sorted donor T cell subpopulations show that donor T cells that bind host I-A antigens preferentially cooperate with nonimmune host B cells. Donor T cells that do not bind detectable amounts of host I-A antigens preferentially help nonimmune donor B cells. By contrast, donor T cells that either bind or do not bind host I-A antigens display no H-2-restricted interaction and help both donor and host immune B cells. These data reveal that the Ia antigen-binding specificity of distinct functional subpopulations of alloactivated donor T cells regulates their I-region-restricted (self or allo) helper activity for nonimmune B cells but not immune B cells. Furthermore, they suggest that T cell-macrophage and T cell-B cell collaboration is mediated by a complementary anti-Ia:Ia receptor:ligand type of interaction in which the receptor of a T cell binds to the ligand of an antigen-presenting macrophage and/or B cell.

Variable synthesis and expression of E alpha and Ae (E beta) Ia polypeptide chains in mice of different H-2 haplotypes.

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice f the d, k, p, and r haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, E alpha and Ae (E beta). Cells from mice of the b and s haplotypes fail to synthesize E alpha chains but do synthesize Ae chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of the f and q haplotypes fail to synthesize either the Ae or E alpha polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact E alpha: Ae complex are consistent with the theory that both the Ae and E alpha polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics of I-region-controlled functions are discussed.

Biogenesis of membrane-bound and secreted immunoglobulins. II. Two forms of the human alpha chain translated in vitro and processed in vivo as distinct polypeptide chains.

Structural differences between alpha m (ther heavy chain of membrane IgA) and alpha s (the heavy chain of secretory IgA) were investigated. Messenger RNA from the human B lymphoblastoid line 32a.1, expressing both membrane and secretory IgA, was translated in a wheat germ cell-free system, resulting in the synthesis of two primary translation products for the alpha chain, that differed in molecular weight. In vivo pulse and pulse-chase experiments demonstrated that two early biosynthetic forms of the alpha chain were subsequently modified to yield three intracellular forms. As shown by endo-beta-N-acetylglucosaminidase H (endo H) treatment, these forms represent two alpha polypeptide chains, with varying compositions of N-linked oligosaccharides. Of the two forms of the alpha chain remaining after endo H treatment, only the form with the lowest molecular weight was associated with cells after long chase periods. The possible significance of this difference from the results with mu and delta chains is discussed. These results indicate that alpha m is distinguished from an alpha s by a difference in both primary structure and intracellular processing. The functional consequences of this distinction, previously shown for the heavy chain of membrane IgM (micrometer) and heavy chain of secretory IgM (microseconds), may reflect a principle common to the secretory and membrane forms of all immunoglobulin heavy chain classes.

Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with α 2-macroglobulin

The alpha polypeptide chain of the complement protein C3 splits into two fragments of 74 000 and 46 000 apparent mol.wt. under certain conditions used to prepare the protein for SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. The cleavage reaction occurs over a wide range of temperatures and from pH 4.6 to 10.6 in the presence of denaturants such as urea, SDS and guanidine hydrochloride. It is also induced by heat-denaturation of C3 in the absence of chemical denaturants. The reaction occurs only with haemolytically active C3, and is not observed with hydroxylamine-inactivated C3 or with C3b. A similar cleavage of the alpha-chain of complement component C4 occurs under the same conditions, forming fragments of 53 000 and 41 000 apparent mol.wt. This reaction is again specific for haemolytically active C4, and does not occur with C4b or hydroxylamine-inactivated C4. The complement component C5, although structurally similar to C3 and C4, does not undergo a reaction of this type. The characteristics of the denaturation-induced cleavage of C3 and C4 match those described for the 'heat-induced' cleavage of alpha 2-macroglobulin [Harpel, Hayes & Hugli (1979) J. Biol. Chem. 254, 8669-8678]. Cleavage of alpha 2-macroglobulin is also specific for the active form of the protein, and does not occur with chemically inactivated or proteinase-cleaved forms. The unusual conditions and specificity of the peptide-bond cleavage in all three proteins suggest that it is an autolytic process rather than being the result of trace proteinase contamination. The active forms of C3, C4 and alpha 2-macroglobulin have the transient ability to form covalent bonds after activation. The autolytic cleavage reaction is likely to be related to the covalent-bond-forming reactions of these proteins.

Evidence for presence of an internal thiolester bond in third component of human complement.

Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.

The effects of mild reduction on the structure and function of C3.

Reduction and alkylation of C3 in nondenaturing buffers by 2 mM DTT (1 hr, 23 degrees C) (C3 R/A) results in a > 99% loss of C3 hemolytic activity. In contrast, sham alkylated protein, or protein allowed to reoxidize from the mildly reduced state, showed full activity. The loss in lytic activity correlated with the alkylation of approximately 6 liberated sulfhydryls, all of which arose from intrachain disulfides in the alpha polypeptide chain of C3. Data from CD experiments indicate that these bonds are essential for maintaining the native conformation of the protein. The loss of hemolytic activity of C3 R/A was neither due to its inability to be cleaved by C3 convertases nor to a dysfunction of the cleavage-induced labile membrane binding site as cells bearing C3 convertases firmly bound C3 R/A to an extent 80% that of native C3. The hemolytic defect in C3 R/A was found to result from its inability, once deposited on C42-bearing cells, to bind C5 and thereby participate in the formation of a C5 convertase. Unlike the effect on hemolytic activity, C3 R/A showed only a partial loss (3-fold) in its ability to bind to C3 receptors on monocytes. Taken together, these studies differentiate in molecular terms several of the functional sites in C3 and stress the significance of intra-alpha-chain disulfides in maintaining the conformation in activated C3 required for binding of C5.

A genetic and biochemical analysis of a polymorphism of bovine alpha S2-casein.

Using gel electrophoresis a genetic polymorphism of alpha S2-casein (Cn) was discovered in individual milk samples from 2 bovine breeds of the eastern part of France (Vosgienne and Montbéliarde). The 3 observed phenotypes (Plate 1) are determined by 2 co-dominant alleles at an autosomal locus. The alpha S2-Cn A variant was the only one known up to now in European breeds (reference variant) and alpha S2-Cn D is a new variant, whose bands overlap the beta-casein A band at pH 8.6, and migrate faster than alpha S2-Cn A at pH 3.0. The sequence of the polypeptide chain alpha S2-Cn D differs from that of alpha S2-Cn A by the deletion of a very acidic nonapeptide, which includes a cluster of 3 phosphoseryl residues. Due to the characteristics of the reference sequence, this deletion cannot be exactly located but it involves residues 50-58, or 51-59, or 52-60. A genetic analysis shows that locus alpha S2-Cn is closely linked to the cluster alpha S1-Cn--beta-Cn--kappa-Cn. The 4 casein species are thus synthesized by 4 closely linked loci.

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the European hedgehog, Erinaceus europaeus.

Globin prepared from hemoglobin of the European hedgehog (Erinaceus europaeus) was separated into alpha and beta polypeptide chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and the resulting peptides were isolated. The sequences of all the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the European hedgehog thus obtained with those of adult hemoglobin of the tupai (Tupaia glis), 35 amino acids substitutions in the alpha chains and 30 in the beta chains were recognized.

Amino Acid Sequences of the α and β Chains of Adult Hemoglobin of the Tupai, <italic>Tupaia glis</italic>

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.

A new hybrid haemoglobin: haemoglobin strumica/beograd occurring in an individual with four haemoglobins

An investigation of the cord blood from full term twin infants revealed an additional haemoglobin F component due to an abnormal alpha chain. The father of the twins, whose blood picture was normal, was shown to have normal alpha and beta polypeptide chains together with variant alpha and beta polypeptide chains. Electrophoresis showed that he had four major haemoglobin components. Separation of the haemoglobin fractions by column chromatography, globin preparation, chain separation, tryptic and chymotryptic digestion and peptide map preparation led to the identification of haemaglobin A, haemoglobin Strumica (alpha2 112His leads to Arg beta2), haemoglobin D Beograd (alpha2beta2 121 Glu leads to Val) and a hybrid haemoglobin Strumica D/Beograd (alpha2 112His leads to Argbeta2 121Glu leads to Val).

Specific enrichment of antigen-binding receptors from sensitized murine lymphocytes.

Splenic T and B lymphocytes from sensitized mice were adsorbed to antigen-coated nylon discs and realesed from the discs by temperature shift as described by Kiefer, H., (Eur. J. Immunol. 1973. 3: 181 and 1975. 5:624). After cell release, lymphocyte-derived antigen-binding material could be recovered from the discs. A major fraction of the activity (70 -80%) binds to anti-immunoglobulin immunosorbents. A minor fraction does not detectably cross-react with gamma, mu, alpha and kappa immunoglobulin poly-peptide chains. When filtrated through Sephadex G-200, the bulk of activity of both fractions elutes in the region of 7 S serum antibody. However, as demonstrated for (4-hydroxy-3-nitro-phenyl)acetyl (NP)-binding material from C57BL/6 lymphocytes, the average affinity for antigen of the two fractions is drastically lower than that of humoral antibody, and the average affinity of the minor fraction is lower than that of the major fraction. The fraction of lymphocyte-derived antigen-binding material that does not adsorb to insolubilized anti-immunoglobulin serum was found to be proportional to the fraction of T lymphocytes in the input cell population and may therefore represent antigen-binding T lymphocyte surface receptors.

Ribonucleoside diphosphate reductase induced by bacteriophage T4. III. Isolation and characterization of proteins B1 and B2.

Ribonucleoside diphosphate reductase determined by bacteriophage T4 consists of a tight complex (alpha2beta2) of the polypeptide chains alpha (Mr = 80,000 to 85,000) and beta (Mr = 35,000). The alpha2 dimer (= protein B1) was purified from Escherichia coli B infected with T4 mutant nrdB55 (Yeh, Y.C., and Tessman, I. (1972) Virology 47, 767-772) which carries an amber mutation in the gene coding for the beta polypeptide chain. Protein B1 contained binding sites for dATP, an allosteric effector of the reductase. The beta2 dimer (= protein B2) was purified by selective desorption with 1 M guanidine HCl from a dATP-Sepharose affinity column containing adsorbed native T4 ribonucleotide reductase. Protein B2, isolated this way, was enzymatically inactive due to partial loss of its iron but it could be reactivated by treatment with ferrous iron. Active protein B2 contained two atoms of non-heme iron per molecule and exhibited the optical and electron spin resonance spectra previously demonstrated in the native enzyme. The T4-induced proteins B1 and B2 were unable to reduce ribonucleotides when assayed separately but were active in combination. The proteins did not form catalytically functional hybrids with proteins B1 and B2 of Escherichia coli ribonucleotide reductase, neither did they cross-react immunologically with the latter. 5-Hydroxymethyl-dCTP, at concentrations above 10 muM, was a positive allosteric effector of T4 ribonucleotide reductase promoting the reduction of the pyrimidine ribonucleotides CDP and UDP. The nucleotide had little effect on E. coli ribonucleotide reductase.

Isolation and analysis of the mechanism of action of an inactivator of C4b in normal human serum.

A complement regulatory principle, C4b inactivator, was isolated in a partially purified form from normal human serum. The C4b inactivator, a beta1-globulin with an approximate mol wt of 88,000 daltons, and which may be identical to C3b inactivator, cleaved C4b in free solution or on the surface of cells and rendered it unable to participate in hemolytic reactions or to interact with cells, having receptors for C4b. C/b inactivator functioned by cleaving the alpha-polypeptide chain of C4b at a single site which was sufficient to dissociate the molecule into two fragments, C4c and C4d, and to inactivate it biological function. Certain structural correlates of C4 functions deriving from these studies are discussed and a model for C4 structure based on these findings is presented.

Stimulation of alpha and beta polypeptide chain synthesis in cultured human marrow by erythropoietin.

Erythropoietin (ESF) acts on erythroid progenitor cells inducing their differentiation into definitive erythroblasts (1, 2). Associated with this ESF effect there is, in many species, an increase in the rate of heme synthesis (2–5). In addition, in lower mammals, both in vivo and in vitro, the hormone has been shown to increase the rate of globin synthesis (6). There is also evidence that ESF stimulates globin formation in cultured human marrow cells (7,8). Further studies on the mechanism of action of ESF have shown that in cultured rodent marrow cells the increase of hemoglobin synthesis by ESF is preceded by an increase in the formation of 9S RNA which may represent globin polypeptide messenger (9). Indirect support for these results has been the increase in production of alpha, beta and gamma globin polypeptide chains by ESF in cultured neonatal calf marrow cells (10). The present studies were undertaken to define further the mechanism of action of ESF on human marrow cells growing in primary culture. The data obtained indicate that associated with the increase in the rate of heme and globin synthesis (8) ESF causes a comparable increase in the rate of synthesis of both the alpha and beta polypeptide chains of globin in these cultured marrow cells. Materials and Method. Culture of bone marrow cells. Human bone marrow cells were obtained by sternal aspiration in patients undergoing this procedure as part of an evaluation for possible metastatic pulmonary carcinoma. Only morphologically normal aspirates were used. The aspirated marrow cells were immediately placed in cold NCTC 109 solution and cultured by the method of Krantz (4). In brief, bone marrow cells were washed twice with cold NCTC 109 solution and then suspended in an incubation medium consisting of 60% NCTC 109 solution, 40% heat-inactivated human plasma of AB type and 50 units/ml of penicillin so as to obtain a cell concentration of 2000 nucleated cells (approx 25% erythroid)/mm of medium.

Alpha-chain disease: a new immunoglobulin abnormality.

A new type of pathological immunoglobulin was found in the serum, urine, and saliva of a young Arab patient with abdominal lymphoma and diffuse lymphoplasmacytic infiltration of the small intestine. This protein is devoid of light chains and is closely related to the alpha polypeptide chains of the gamma(A1) (Le) subclass of immunoglobulin A. It is characterized by electrophoretic heterogeneity, tendency toward polymerization, and a high carbohydrate content. No intracellular synthesis of light chain was detected.

In vivo and in vitro interactions of human haemoglobins A,S and C with a variant haemoglobin E.

THE predominant haemoglobin in the blood of most human adults is type A, a tetrameric haemo-protein composed of two pairs of identical polypeptide chains—alpha and beta chains—inherited as nonallelic autosomal dominant traits1. I have found a family of African Negro origin which possesses four different major haemoglobins (Fig. 1): Hb A, S, C and E. These were identified preliminarily by their characteristic electrophoretic mobilities in starch gel at pH 8.6 (Fig. 1)2; the appearance of Hb C or E on starch was verified by agar gel electrophoresis at pH. 6.2 (Fig. 1)3, and appearance of Hb S was verified by deoxygenation of samples of whole blood with sodium metabisulphite : this causes Hb S to polymerize, distorting the erythrocytes into rigid, elongate forms known as sickle cells4,5.

Hemoglobin M Reserve: studies on identification and characterization.

Abstract A family with hereditary cyanosis secondary to a hemoglobin M is reported; pending completion of amino acid analysis this abnormal hemoglobin is designated hemoglobin M Reserve . The M hemoglobin was separated by electrophoretic and chromatographic techniques and comprised 30 per cent of the proband's total hemoglobin. A qualitatively abnormal A 2 hemoglobin was also demonstrated. The isolated hemoglobin M had spectral absorption abnormalities in the 480 to 520 and 600 to 680 m μ ranges when in the oxy-, deoxy-, met-, and cyanmethemoglobin states; however, prolonged reduction with dithionite eliminated the spectral abnormalities present in the deoxygenated state. The structural abnormality of the hemoglobin M molecule was located in the alpha polypeptide chain and resulted in reduced oxygen affinity and decreased reversible oxygen binding capacity. Other demonstrable abnormalities included increased resistance to nitrite-induced methemoglobin formation, increased rate of ferricyanide-induced methemoglobin formation, and an increased rate of methemoglobin formation duing hemoglobin autooxidation. There were no demonstrable abnormalities in red cell content of reduced glutathione, glutathione peroxidase, TPN-methemoglobin reductase, or DPN diaphorase to explain these changes in rates of heme iron oxidation. The behavioral abnormalities are apparently secondary to the molecular conformational changes within the hemoglobin M molecule that are consequent upon the amino acid substitution, internal bonding of the ionized radical of the abnormal amino acid with heme iron, and altered interaction between alpha and beta polypepttde chains.